JP3333969B2 - D-Ketohexose-3-epimerase, its production method and use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to D-ketohexose.
The present invention relates to 3-epimerase, its production method and use.

[0002]

BACKGROUND OF THE INVENTION Epimerase is known as Enzyme Nomenclature.
(Academic Press, I, United States
nc. 1992) is known to act on various carbohydrates. However, epimerases known to date include, for example, ribulose phosphate · 3
-Epimerase (EC 5.1.3.1) and UDP-glucose 4-epimerase (EC 5.1.3.2)
It acts mainly on phosphorylated carbohydrates and carbohydrates bound to UDP and the like, and cannot be used in industrial applications for producing free neutral carbohydrates. On the other hand, epimerase which acts on free carbohydrates has two examples which act on aldose, namely, aldose 1-epimerase (EC 5.1.3.3) and cellopiose epimerase (EC 5.1.3.11). ) Is only known. The former is an enzyme that catalyzes epimerization between the α- and β-anomers at position 1 of aldose, and the latter is an enzyme that catalyzes between the α and β anomers of cellobiose. Although epimerase acting on free ketose is expected, its existence is not yet known.

[0003]

SUMMARY OF THE INVENTION It is desired to establish a ketose epimerase which acts on free ketose, a method for producing the same, and a use thereof.

[0004]

Means for Solving the Problems The present inventors have proposed a ketoheki.
Between source over scan, or between ketopentose easily by focusing on epimerase capable of epimerization remains free carbohydrates,
I have been searching for this enzyme. As a result, a new D-
A method for producing D-ketohexose, D- or L-ketopentose using the enzyme, a method for producing converted ketoose, and a method for producing ketohexose 3-epimerase. The present invention was completed by establishing a method for producing a sweetener containing ketose. That is, the D-ketohexose-3 of the present invention
-Epimerase is a novel enzyme having an activity of epimerizing D-ketohexose at position 3 to produce the corresponding D-ketohexose, and has the following physicochemical properties.

(1) Action and Substrate Specificity The D-ketohexose at position 3 is epimerized and the corresponding D
-Produce ketohexose. Epimerize the 3-position of D- or L-ketopentose to the corresponding D- or L-
Generate ketopentose.

(2) Optimum pH and pH stability It has an optimum pH of pH 7 to 10, and is stable at pH 5 to 10.

(3) Optimum temperature and thermal stability: Optimum temperature around 60 ° C, stable at 50 ° C or less.

(4) Ultraviolet absorption spectrum An absorption band is shown at 275 to 280 nm.

The D-ketohexose-3-epimerase of the present invention may be produced by culturing a microorganism capable of producing D-ketohexose-3-epimerase to produce D-ketohexose-3-epimerase.

[0010] As the microorganism, for example, a bacterium belonging to the genus Pseudomonas is suitable.
No. 266996 discloses Pseudomonas.
Chicory ST-24 (FERM BP-2736) and its mutants can be advantageously used.

According to a conventional method, the cells are cultured in a nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, a vitamin, etc. for about 1 to 5 days, preferably in a liquid medium under aerobic conditions by aeration and stirring. And D-ketohexose-3-epimerase may be collected from the resulting cells or cultures such as culture supernatants. Usually, the culture can be used as crude D-ketohexose-3-epimerase. If necessary, filter, centrifuge, salt out,
It can be partially purified, collected, and used by a known method such as dialysis, concentration, and freeze-drying. When a higher degree of purification is required, for example, adsorption / elution to an ion exchanger,
It is optional to use the gel with the highest purity by combining gel filtration, isoelectric focusing, electrophoresis, high performance liquid chromatography, affinity chromatography, and adsorption / elution to a monoclonal antibody.

The conversion reaction of the present invention, that is, D-
From at least one kind of ketoose selected from ketohexose, D-ketopentose, and L-ketopentose, the 3-position of the ketoose is epimerized, and the corresponding D-ketohexose,
In a conversion reaction for producing D-ketopentose and L-ketopentose, the D-ketohexose-3-epimerase of the present invention can be immobilized by a known method and used repeatedly for the reaction or used for a continuous reaction. It can be implemented advantageously.

This conversion reaction is usually carried out under the following conditions. Substrate immersion is 1 to 60 w / v%, preferably about 5 to 50 w / v%, reaction temperature is 10 to 70 ° C, preferably about 30 to 60 ° C, and reaction pH is 5 to 10, preferably about 10 7-10, enzyme activity is 1 per gram of substrate
It is selected from a unit or more, preferably from 50 to 5,000 units. The reaction time can be appropriately selected, but in the case of a batch reaction, it is usually 5 to 50 in view of economy.
A time range is chosen.

[0014] In this way, the reaction solvent solution was converted is contained and ketose newly generated and the raw material of the ketose, which is directly sweeteners, humectants, crystallization inhibitors,
It can be advantageously used as a shine imparting agent. Usually, the reaction solution is decolorized using activated carbon, desalted and concentrated using H-type and OH-type ion exchange resins according to a conventional method to obtain a syrup-like product.

If necessary, the concentrate is separated and purified from the newly formed ketose and the starting ketose by column chromatography using, for example, an alkali metal type or alkaline earth metal type strongly acidic cation exchange resin. It is advantageous to concentrate the newly formed ketose-rich fraction to obtain a syrup-like product, and in the case of a crystallizable ketose, to crystallize to obtain a crystalline product. It is also advantageous to use the separated ketose of the raw material again as the raw material for the conversion reaction.

The ketose thus obtained is suitable as a sweetener, and can be used as a food, drink, feed, feed, toothpaste,
Sweetening of oral ingestion such as oral balm, sublingual tablet, oral medicine,
It can be used advantageously to improve palatability. In addition, it can be advantageously used as a carbon source for fermentation, a reagent, a raw material for chemicals and pharmaceuticals, an intermediate, and the like.

Hereinafter, a few embodiments of the present invention will be described.

[0018]

Example 1 Ammonium sulfate 0.2 w / v%, potassium potassium phosphate 0.24 w / v%, dipotassium phosphate 0.5
6w / v%, magnesium sulfate heptahydrate 0.01w / v
%, Yeast extract 0.5 w / v%, D-glucose 1 w /
v% and deionized water in a jar fermenter and sterilized at 120 ° C. for 20 minutes, and then Pseudomonas chicory ST-24 (FERM BP-273).
The seed culture solution (1 v / v%) of 6) was aseptically added and cultured at 30 ° C. for 40 hours with aeration and stirring. The obtained culture solution 8
The cells were collected by centrifugation from 0 l, triturated with activated alumina, and 50 mM Tris-HCl buffer (pH 7.0).
The enzyme was extracted in 5).

This crude enzyme solution was purified by repeatedly performing a fractionation precipitation method using polyethylene glycol 6000 (hereinafter abbreviated as PEG) in the presence of manganese chloride. That is, 0.1M
PEG 5 w / v% and 18 w / v% in the presence of manganese chloride
Was dissolved in the same buffer. This PE
The G fraction was purified twice. Then at 50 ° C for 20
After denatured protein was removed by centrifugation after a heat treatment for 5 minutes, the protein was adsorbed on DEAE-Toyopearl 650M and purified by elution with potassium chloride. After desalting and concentration by ultrafiltration (filtration membrane using Toyo filter paper UK-10), it was further purified by a gel filtration method using Sephadex G150 (Pharmacia, Sweden). The active fraction was concentrated and further purified by focal electrophoresis using Amforine (LKB, Sweden).

The activity of the present enzyme was measured as follows. That is, the composition of the reaction solution is 50 mM Tris-HCl buffer (pH
7.5) 50 μl of 100 μl, 40 mM D-tagatose
μl and 50 μl of enzyme solution. The reaction is 60
Min Magyo not the D- sorbose The product was determined by HPLC. One unit of the enzymatic activity was defined as the amount of the enzyme capable of epimerizing 1 μmol of D-tagatose per minute to produce D-sorbose.

The purification process is summarized in Table 1.

[0022]

[Table 1]

As is clear from the results in Table 1, the specific activity was improved by about 290 times by this purification process, and the activity recovery at this time was about 2%. When examined using this purified enzyme, the physicochemical properties of the D-ketohexose-3-epimerase of the present invention are as follows.

[0024] (1) action and of substrate specificity D- and L- Ketoheki source over scan eight, D- Ketoheki
Source over scan four (D- tagatose, D- sorbose, D- fructose, D- psicose) acts on all the respective position 3 epimerization, converted to the corresponding D- Ketoheki source. It also acts on all four D- and L-ketopentoses (D- and L-xylulose, D- and L-ribulose), epimerizes the respective 3-position and converts it to the corresponding D- or L-ketopentose. .

The present enzyme most strongly catalyzes the conversion reaction between D-tagatose and D-sorbose, and the conversion reaction between D-fructose and D-psicose is the reaction between D-tagatose and D-sorbose. It shows about 30-40% activity, and furthermore, the conversion reaction between D- or L-ketopentose shows several% activity between D-tagatose and D-sorbose. These reactions are reversible and equilibrium reactions. These reactions do not require coenzymes or metal ions.

(2) Optimum pH and pH stability The optimum pH was measured according to the activity measurement method. Optimum pH
1 are shown in FIG. In FIG. 1, the symbol-□-is 5
0 mM citrate buffer was used.
0 mM maleate buffer, symbol -−- is 50 m
M Tris-HCl buffer, symbol-●-indicates 50 mM
2 shows a glycine-NaOH buffer. As is clear from FIG. 1, pH 7 to 10 is the optimum pH.

[0027] The pH stability is as follows.
The activity after maintaining at 0 ° C. for 60 minutes was measured and examined. The results are shown in FIG. As is clear from FIG. 2, pH 5 to 10 is stable.

(3) Optimum temperature and thermal stability The optimum temperature was measured according to the activity measuring method. Result is,
As shown in FIG. 3, the optimum temperature is around 60 ° C. The thermostability was determined by measuring the activity after keeping the enzyme in 50 mM Tris-HCl buffer (pH 7.5) for 10 minutes at various temperatures. The results are stable below 50 ° C., as shown in FIG.

(4) Ultraviolet absorption spectrum An absorption band is shown at 275 to 280 nm.

(5) Molecular weight The molecular weight was measured using a column (1.2 × 100 cm) packed with Sephadex G150, using 25 mM Tris-HC.
1 buffer (pH 7.5) as eluent, flow rate 0.15m
It was determined by gel filtration chromatography under the condition of 1 / min. The results are shown in FIG. In FIG. 5, symbol A represents bovine serum albumin (BSA) having a molecular weight of 67,000.
, The symbol B is ovalbumin having a molecular weight of 45,000,
Symbol C indicates the present enzyme, symbol D indicates chymotrypsinogen A having a molecular weight of 25,000, and symbol E indicates cytochrome C having a molecular weight of 12,500. As is clear from FIG. 5, the molecular weight of the present enzyme (C) is 41,000 ± from the standard curve prepared with various proteins (A, B, D, E) of known molecular weights.
3,000.

(6) Isoelectric point When determined by a focal electrophoresis method using an ampholine (manufactured by LKB, Sweden), pH 4.3 ±
0.2.

(7) Polyacrylamide gel electrophoresis The results of polyacrylamide gel electrophoresis of the present enzyme are shown in FIG. In FIG. 6, symbol I indicates the starting point of electrophoresis, symbol II indicates the present enzyme, and symbol III indicates bromophenol blue. As is clear from FIG. 6, the present enzyme shows a single band.

[0033]

Example 2 Conversion reaction between D-tagatose and D-sorbose 5% w / v aqueous solution of D-tagatose (pH
In 7.5), the enzyme solution partially purified by the method of Example 1 until the second PEG fractionation was added to 500-g / D-tagatose gram.
The reaction was added at a unit rate and reacted at 40 ° C. for 30 hours. After the reaction, the reaction solution is decolorized using activated carbon according to a conventional method, and then Diaion SK1B (H type, trade name manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) and Diaion WA30 (OH
The mixture was desalted using a mold (trade name, manufactured by Mitsubishi Chemical Industry Co., Ltd.) and concentrated under reduced pressure to obtain a transparent syrup containing D-tagatose and D-sorbose at a concentration of about 60%. Then, separation and purification were performed by column chromatography using Dowex 50W-X4 (salt type strongly acidic cation exchange resin, trade name manufactured by Dow Chemical Company), and further concentrated, crystallized and separated to obtain crystalline D-sorbose. Obtained in about 55% yield. Examining the physicochemical properties of this product, The infrared absorption spectrum (this product is shown in FIG. 7 and the reagent D-sorbose as a standard substance is shown in FIG. 8) are all in good agreement with those of the standard D-sorbose. From these results, conversion from D-tagatose by this enzyme,
The produced saccharide is determined to be D-sorbose. The product can be advantageously used as a sweetener, a carbon source for fermentation, a reagent, a raw material or an intermediate of a chemical or pharmaceutical. In addition, since this enzyme reaction is a reversible reaction, D-
By using sorbose, it is easy to collect D-tagatose as a product.

[0034]

Example 3 Conversion reaction between D-fructose and D-psicose Enzyme solution partially purified to a DEAE-Toyopearl step by the method of Example 1 in a 10 w / v% aqueous solution of D-fructose (pH 7.5) At a rate of 1,500 units per gram of D-fructose.
Allowed to react for hours. After the reaction, the reaction solution was treated in the same manner as in Example 2,
Decolorization, desalting, and concentration under reduced pressure gave a transparent syrup containing D-psicose. This syrup was subjected to column chromatography using a salt type strongly acidic cation exchange resin in the same manner as in Example 2, separated and purified, and concentrated to obtain a syrup-like D-psicose at a yield of about 25% based on solids. The physicochemical properties of this product were in good agreement with those of standard D-psicose. The product can be advantageously used as a sweetener, a carbon source for acid fermentation, a reagent, a raw material / intermediate of a chemical / pharmaceutical, and the like. Further, since the present enzyme reaction is a reversible reaction, it is easy to collect D-fructose as a product by using D-psicose as a raw material.

[0035]

Example 4 Conversion reaction between D-fructose and D-psicose An enzyme partially purified by a method of Example 1 up to the second round of PEG fractionation in a 10 w / v% aqueous solution of D-fructose (pH 7.0) The liquid was added at a rate of 1,000 units per gram of D-fructose, and reacted at 50 ° C. for 30 hours. After the reaction, the reaction solution was decolorized, desalted and concentrated under reduced pressure in the same manner as in Example 2 to obtain a transparent syrup containing D-fructose and D-psicose at a yield of about 90 per solid.
%. This product is suitable as an elegant high-intensity sweetener and can be advantageously used not only as a sweetener for various foods and drinks, but also as a humectant, a crystal precipitation inhibitor, a shine imparting agent, and the like.

[0036]

EXAMPLE 5 D- carboxymethyl Le roast 1 w / v% aqueous solution of the conversion reaction D- xylulose between D- ribulose (pH 7.5)
Then, the enzyme solution partially purified to the DEAE-Toyopearl step by the method of Example 1 was used in an amount of 3,0 per D-xylulose gram.
The mixture was added at a rate of 00 units and reacted at 35 ° C. for 50 hours.
After the reaction, the reaction solution was decolorized, desalted and concentrated under reduced pressure in the same manner as in Example 2 to obtain a transparent syrup containing D-ribulose. By column chromatography using a similarly strong-acid cation exchange resin of the present syrup as in Example 2, to give the a syrupy D- ribulose was obtained in about 20% dsb yield and concentrated. The physicochemical properties of this product were in good agreement with those of standard D-ribulose. The product can be advantageously used as a sweetener, a carbon source for fermentation, a reagent, a raw material for chemicals and pharmaceuticals, an intermediate, and the like. In addition, since the present enzyme reaction is a reversible reaction, it is easy to collect D-xylulose as a product by using D-ribulose as a raw material.

[0037]

Example 6 Conversion reaction between L-xylulose and L-ribulose The enzyme solution partially purified to the DEAE-Toyopearl step by the method of Example 1 was applied to a 1 w / v% L-xylulose aqueous solution (pH 7.5). , L-xylulose at a rate of 3,000 units per gram, and reacted at 35 ° C. for 50 hours. After the reaction, the reaction solution was decolorized and treated in the same manner as in Example 2.
After desalting and concentration under reduced pressure, a clear syrup containing L-ribulose was obtained. This syrup was separated and purified by column chromatography using a salt type strongly acidic cation exchange resin in the same manner as in Example 2, and concentrated to obtain a syrupy L-ribulose at a yield of about 20% per solid. The physicochemical properties of this product were in good agreement with those of standard L-ribulose. The product can be advantageously used as a sweetener, a carbon source for fermentation, a reagent, a raw material and an intermediate for chemicals and pharmaceuticals, and the like. Also,
Since this enzyme reaction is a reversible reaction, it is easy to collect L-xylulose as a product by using L-ribulose as a raw material.

[0038]

The D-ketohexose-3-epimerase of the present invention is free D-ketohexose, D- or L-ketohexose.
-Act on ketopentose to epimerize the 3-position of these ketoses and easily generate the corresponding D-ketohexose, D- or L-ketopentose. This reaction opens the way for mass production of various ketoses, which had been difficult to produce conventionally. Therefore, the establishment of the D-ketohexose-3-epimerase of the present invention, its production method and use is of great industrial significance not only in the sugar industry but also in the food, cosmetics and pharmaceutical industries related thereto.

[Brief description of the drawings]

FIG. 1 is a diagram showing an optimum pH.

FIG. 2 is a diagram showing pH stability.

FIG. 3 is a diagram showing an optimum temperature.

FIG. 4 is a diagram showing thermal stability.

FIG. 5 is a diagram showing the molecular weight of the present enzyme.

FIG. 6 is a photograph of polyacrylamide gel electrophoresis of the present enzyme.

FIG. 7 is a diagram showing an infrared absorption spectrum of D-sorbose produced from D-tagatose according to the present invention.

FIG. 8 is a diagram showing an infrared absorption spectrum of standard D-sorbose.

[Explanation of symbols]

A Bovine serum albumin (BSA) B Ovalbumin C Enzyme (D-ketohexose / 3 epimerase) D Chymotrypsinogen A E Cytochrome C I Electrophoresis start point II Enzyme (D-ketohexose / 3 epimerase) III Bromphenol blue

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 9/00-9/98 C12P 19/00-19/24 BIOSIS (DIALOG) EUROPAT (QUESTEL) JICST file (JOIS) WPI (DIALOG) PubMed

Claims (9)

(57) [Claims] 1. It has the following physicochemical propertiesPseudomona
Can be obtained from bacteria belonging to the genusD-ketohexo
Source 3-epimerase. (1) Action and Substrate Specificity The D-ketohexose at position 3 is epimerized and the corresponding D
-Produce ketohexose. Epimerizing the 3-position of D- or L-ketopentose,
Generate the corresponding D- or L-ketopentose. (2) Optimum pH and pH stability It has an optimum pH of 7 to 10, and is safe at pH 5 to 10.
Fixed. (3) Optimum temperature and thermal stability The optimum temperature is around 60 ° C and stable at 50 ° C or less. (4) Ultraviolet absorption spectrum An absorption band is shown at 275 to 280 nm.(5) Molecular weight 41,000 ± 3,000 (gel filtration chromatography
By ). 2. The D-ketohexose-3 according to claim 1, wherein a bacterium belonging to the genus Pseudomonas and having an ability to produce D-ketohexose-3-epimerase is cultured in a nutrient medium.
2. The D-ketohexose-3-epimerase according to claim 1, which is obtained by producing epimerase and collecting it. 3. A D-ketohexose-3-epimerase-producing bacterium belonging to the genus Pseudomonas , which is cultured in a nutrient medium to produce D-ketohexose-3-epimerase according to claim 1 or 2. 3. The method for producing D-ketohexose-3-epimerase according to claim 1 or 2, wherein 4. The solution according to claim 1 or 2, wherein the solution containing one or more ketoses selected from D-ketohexose, D-ketopentose and L-ketopentose.
A ketoose conversion method, which comprises causing ketohexose-3-epimerase to act to epimerize the 3-position of the ketoose. 5. The method according to claim 4, wherein D-ketohexose-3-epimerase is immobilized. 6. The solution according to claim 1 or 2, wherein the solution containing one or more ketoses selected from D-ketohexose, D-ketopentose and L-ketopentose.
A method for producing ketose, which comprises reacting ketohexose-3-epimerase to epimerize the 3-position of the ketose, producing a corresponding ketose, and collecting the ketose. 7. The method for producing ketose according to claim 6, wherein D-ketohexose-3-epimerase is immobilized. 8. The method according to claim 6, wherein the method for collecting ketose is a method for collecting ketose separated and purified by column chromatography using a salt type strongly acidic cation exchange resin. Production method. 9. The solution according to claim 1 or 2, wherein the solution containing one or more ketoses selected from D-ketohexose, D-ketopentose and L-ketopentose.
A method for producing a ketose-containing sweetener, comprising reacting ketohexose-3-epimerase to epimerize the 3-position of the ketose to produce a corresponding ketose, and collecting the ketose-containing solution.